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Severe little leaf and excessive shoot proliferation symptoms typical of the peanut little leaf (PnLL) disease were observed in different peanut (Arachis hypogaea) fields, variety K-6, at Kadiriand Gooty regions, of Andhra Pradesh, India, during the monsoon season of 2018–19. Disease incidence was 2–3%. The phytoplasma etiology of the recorded disease was confirmed by amplifying ~1.25 kb DNA products of 16S rRNA gene from symptomatic plants using nested polymerase chain reaction with phytoplasma specific primers, P1/P7 and R16F2n/R16R2, respectively. A further confirmation of the phytoplasma etiology was obtained by amplification of 840 bp, 1094 bp and 465 bp DNA fragments using phytoplasma specific primers targeting secA, tuf and SAP11 genes, respectively. Sequence and phylogenetic analyses of the amplified 16S rRNA, secA, tuf and SAP11 genes revealed that the detected phytoplasma is a member of the peanut witches’-broom phytoplasma group or 16SrII group (‘Candidatus Phytoplasma australasia’). Also, on the basis of computer-simulated RFLP (= in silico RFLP) analysis of amplified 16S rRNA gene, the detected phytoplasma was assigned to subgroup D (16SrII-D). Parthenium hysterophorus plants showing witches’ broom and Cleome viscosa plants with little leaf symptoms, both collected in the mentioned peanut fields were also infected by similar strain of phytoplasma which proved to be identical with each of the molecular marker employed to the peanut-infecting agent in India. This is the first report on the association of ‘Ca. P. australasia’ (16SrII-D subgroup phytoplasma) with PnLL disease in India.

Symptoms of decline, leaf yellowing and reddening, little leaf and malformation were observed in apricot, guava, lychee, mango, pomegranate and grapevine in fruit orchards belonging to three states of India namely Delhi, Maharashtra and Jammu & Kashmir during 2016–2017. Phytoplasmas belonging to three different groups were detected in all the symptomatic fruit tree samples in polymerase chain reaction with phytoplasma specific primer pairs amplifying 16S rRNA and secA genes. Pair wise sequence comparison and phylogenetic analysis confirmed the presence of phytoplasmas. A ‘Candidatus Phytoplasma asteris’ - related strain was detected in apricot showing decline symptoms in Siot (Jammu & Kashmir). ‘Ca. P. australasia’ - related strain was identified in guava, lychee, mango and pomegranate in Rajbag, Sunjwan (Jammu & Kashmir), Pusa (Delhi) and Baramati (Maharashtra), respectively, exhibiting little leaf, leaf yellows and malformation symptoms. Furthermore, a rice yellow dwarf group (16SrXI-B) - related strain was detected in pomegranate and grapevine that showed leaf yellowing and reddening at Pusa (Delhi) and Baramati (Maharashtra), respectively. Subgroup analysis using virtual RFLP of 16S rDNA sequences allowed enclosing these phytoplasma strains into 16SrI-B, 16SrII-D and 16SrXI-B subgroups. In the study, identification of phytoplasma subgroups, 16SrI-B in apricot, 16SrII-D in guava, lychee, mango and pomegranate (Pusa) and 16SrXI-B in pomegranate (Baramati) and grapevine, are the first records globally.

Zinnia elegans belonging to the asteraceae family is a flowering annual summer plant and is native to Mexico. Symptoms of excessive leaf curling, little leaf, stunting and witches’ broom were observed on Z. elegans in Tripura state of India during 2016–2017. PCR assays using phytoplasma universal primer pairs P1/P7 and R16F2n/R16R2 and tomato leaf curl virus coat protein specific primers yielded specific amplifications of ~ 1.25 kbp and ~ 800 bp, respectively from the symptomatic zinnia leaf samples. Sequence comparison, phylogenetic and virtual RFLP analysis of 16S rRNA gene sequences of zinnia isolate confirmed the presence of ‘Candidatus Phytoplasma asteris’ (16SrI-B subgroup). Further coat protein sequence comparison and phylogenetic tree analysis of coat protein gene confirmed association of begomovirus with zinnia symptomatic samples and identified it as a strain of tomato leaf curl virus. Our results confirmed a mixed infection of ‘Ca. P. asteris’ and begomovirus with zinnia leaf curl and witches’ broom symptoms.

Symptoms of excessive shoot proliferation were observed in the Njallani cultivar of small cardamom accompanied by stunting of stalks with fewer degenerated capsules at Nedumkandam Panchayat of Idukki district of Kerala in 2017. Five symptomatic Elettaria cardamomum shoot proliferation (ECSP) plant samples were collected and processed for DNA extraction and PCR assays utilizing universal phytoplasma 16S ribosomal-specific primers pair, P1/P7 followed by R16F2n/R16R2. Sequence comparison analysis of the R16F2n/R16R2 region of 16SrRNA gene showed 100% sequence identity with the ‘Candidatus Phytoplasma australasia’- related strain. Phylogeny and virtual RFLP analyses of 16S rRNA gene sequences confirmed the association of ‘Ca. P. australasia’ strain subgroup D with ECSP disease. The association of 16SrII group was further established and validated by amplifying phytoplasma-specific multilocus candidate genes by utilizing specific primers of secA, secY, SAP11, and tuf genes. The multilocus gene sequence comparison analysis again confirmed the association of ‘Ca. P. australasia’ with the ECSP phytoplasma isolate. This is the first report of phytoplasma association with small cardamom. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13205-019-1944-9) contains supplementary material, which is available to authorized users.

During a survey performed in sapota orchards of India, from 2015 to 2018, symptoms of phyllody, little leaf, flat stem and witches’ broom were observed in three states: Karnataka, Kerala and Tripura. The association of phytoplasmas was confirmed in all the symptomatic sapota samples by using nested PCR specific primers (P1/P7, R16F2n/R16R2 and 3Far/3Rev) with amplification of fragments of ~ 1.25 kb and ~ 1.3 kb. Association of three phytoplasma groups, aster yellows with flat stem from Tripura (Lembucherra), clover proliferation with phyllody symptoms at Karnataka (Bengaluru) and bermuda grass white leaf with flat stem and little leaf from Kerala (Thiruvananthapuram) and Tripura (Cocotilla) were confirmed by 16S rRNA gene sequence comparison analysis. Virtual RFLP analysis of 16S rRNA gene sequences using pDRAW32 further classified the sapota phytoplasma isolates into 16SrI-B, 16SrVI-D and 16SrXIV-A subgroups. This is the first report on identification of three phytoplasma groups in sapota in world.

Cashew nut (Anacardium occidentale L.), an evergreen perennial tree, is an economically important cash crop extensively used in many Indian desserts and cuisines. Cashew nuts are used for their nutritional value and are a major source of protein, fats, vitamins, oleic acid, and palmitic acid. Cashew nut cultivation is undertaken in 17 states of India covering an area of 1.04 Mha. India is the second-largest cashew nut producer in the world, producing about 817,000 MT of raw cashew nuts per annum (https://cashew.icar.gov.in). During a survey in June 2018 of cashew nut orchards in Nagicherra (latitude N 23° 48′ 37.6″; longitude E 91° 19′ 29.7″), West Tripura, India, cashew nut plants were found exhibiting deformed nuts and inflorescence decline (CaID) with prevalence of 5.6% (in a 1-ha area), which were suggestive of phytoplasma infection. Three symptomatic and asymptomatic cashew nut samples were collected, and DNA was extracted from tender shoots using CTAB. To detect the association of phytoplasma with symptomatic cashew nut samples, the isolated genomic DNA samples were assayed for amplification of 16S rRNA region using phytoplasma-specific primer pairs, namely, P1/P7 followed by nested PCR primers R16F2n/R16R2. The 16S rRNA gene of phytoplasma was detected in all collected symptomatic CaID samples, but no amplification was observed in asymptomatic samples. The obtained nested PCR fragments of ∼1.25 kb were sequenced directly (MK076296, MK729150) which showed 99.8% sequence identity with ‘Candidatus Phytoplasma australasia’, strain ‘Carica papaya’ (Y10097). The virtual restriction fragment length polymorphism analysis of 16S rRNA gene sequences delineated the CaID strain to 16SrII-D subgroup with a similarity coefficient of 1.0 using iPhyClassifier. The association of CaID phytoplasma strain with symptomatic cashew nut samples was further established by amplifying phytoplasma-specific partial secA gene (Hodgetts et al. 2008) and tuf gene (Al-Subhi et al. 2018). The phylogenetic analysis of the obtained sequences of 16S rRNA, secA (MK726365, MK726366), and tuf (MK726367, MK726368) genes confirmed the close association of CaID phytoplasma isolates with ‘Ca. P. australasia’ 16SrII-D subgroup member strains. To the best of our knowledge, this is the first report of the association of ‘Ca. P. australasia’ (16SrII-D) with inflorescence decline symptoms of cashew nut in the world. This is significant because phytoplasma strains of 16SrII-C and D have widespread host range including several fruit trees all across India (Rao et al. 2017). These strains are also known to be associated with diseases in weeds, acting as alternate hosts, spreading phytoplasmas through polyphagous insect vectors (Thorat et al. 2016). Likewise, the cashew plants are intercropped in coconut plantations and with plants like maize, groundnut, cowpea, millets, pineapple, and cassava, which are known to be susceptible to phytoplasma-related diseases, posing a threat to cashew nut plantations (Rao et al. 2017; Visalakshi et al. 2015).

Symptoms of mango malformation and witches’ broom were observed in mango orchards at Ludhiana, Punjab, India during 2020. Phytoplasmas were detected in the symptomatic mango samples by polymerase chain reaction with specific primer pairs amplifying 16S rRNA and secA genes. Pair wise sequence comparison and phylogenetic analysis of 16S rRNA and secA gene sequences of mango phytoplasma strains indicate their relatedness with phytoplasmas in the aster yellows group. In silico RFLP analyses using the iPhy Classifier of the 16S rDNA sequence of the mango phytoplasma allowed its classification into the 16SrI-D subgroup. This is first report of this phytoplasma in mango plants showing a malformation and witches’ broom disease.

Leaf yellowing symptoms were observed on Acacia mangium in the Sipahijala district of Tripura, India, during June 2017. Symptomatic and asymptomatic leaf samples (three of each) were collected from roadside trees of A. mangium for DNA extraction using the CTAB method. Amplicons of ~1.25 kb and ~480 bp were detected in all the symptomatic samples using the phytoplasma‐specific universal 16S rRNA and secA gene primers. Pair wise sequence analysis of 16S rRNA gene sequences, virtual RFLP and phylogenetic analysis revealed that the phytoplasma strain associated with A. mangium belonged to phytoplasma subgroup 16SrII‐C. This is the first report of an association between the 16SrII‐C subgroup and A. mangium leaf yellowing.

Ziziphus oenoplia, also known as jackal jujube or small fruited jujube is a spiny, evergreen small tropical fruit tree. During a survey in October-November 2019, the jujube trees exhibited severe witches’ broom symptoms, therefore, an investigation was carried out, to verify the presence of phytoplasmas in these symptomatic plants. Total DNA was extracted from leaves of jujube plants using a CTAB method and a PCR assay was performed using 16S rRNA gene specific primer pairs P1/P7 and 3Far/3Rev. Amplicons of about 1.3 kb were consistently amplified from all the symptomatic plant samples in nested PCR assays, while, no amplification was observed in non-symptomatic samples. The phylogenetic analysis of 16Sr DNA sequence of the detected phytoplasma strain indicates its clustering with ‘Candidatus Phytoplasma balanitae’ strains and also its close relatedness to 16SrV-F subgroup phytoplasmas. The virtual RFLP analysis of the 16Sr DNA sequence revealed that it could tentatively be classified into a novel phytoplasma subgroup since its AluI restriction profile was unique to earlier reported subgroup strains in 16SrV group.

Phytoplasma suspected symptoms of little leaf, chlorosis, yellowing, phyllody, witches’ broom, flat stem, flower virescence and stunting were observed in sixteen plant species during surveys in fields, gardens, fruit orchards and nurseries of six districts of Tripura, India in 2017–2018. Out of 15 symptomatic plant samples belonging to different species, 12 were positive through nested PCR assays for phytoplasma presence. Pair wise sequence comparison, phylogeny and virtual RFLP analysis of phytoplasma partial 16S rDNA sequences of these 12 samples indicated the presence of different phytoplasma groups and subgroups (16SrI-B, 16SrII-D, 16SrVI-D, 16SrXIV-A 16SrI, 16SrIX and 16SrXI). ‘Candidatus Phytoplasma asteris’-related phytoplasmas (16SrI-B) were identified in sesame phyllody affected plants at South Tripura and in crape jasmine, Celosia argentea and Zinnia elegans from Unakoti, Dhalai, North Tripura and West Tripura district. In West Tripura ‘Ca. P. australasiae'related phytoplasmas (16SrII-D) were identified with Jatropha hastata and bitter gourd; ‘Ca. P. trifolii’-related phytoplasmas (16SrVI-D) were identified in chili and brinjal and ‘Ca. P. cynodontis’-related phytoplasmas (16SrXIV-A) were identified in palm grass samples from Sipahijala district. ‘Ca. P. phoenicium’-related phytoplasmas (16SrIX) were identified in cowpea; ‘Ca. P. oryzae’-related phytoplasmas were identified in arecanut from North Tripura and ‘Ca. P. asteris’-related phytoplasmas were detected in Toona ciliata from Jampui Hills. This study suggests the presence of a wide genetic diversity of phytoplasmas infecting different crops and weeds in the Tripura state of India.